Transfusion transmissible pathogens are prevalent in healthy cats eligible to become blood donors.

OBJECTIVES: This study aims to determine the prevalence of subclinical infectious agents considered core pathogens for worldwide screening in healthy, client-owned, indoor cats eligible to become blood donors in Spain and Portugal. MATERIALS AND METHODS: Blood samples of healthy, indoor, domestic cats selected to be potential blood donors were tested for feline leukaemia virus antigens, feline immunodeficiency virus antibodies and polymerase chain reactions for Mycoplasma haemofelis, Candidatus Mycoplasma haemominutum, Candidatus Mycoplasma turicensis, feline leukaemia virus provirus, Leishmania spp. and Bartonella spp. Not all donors were tested for all agents. RESULTS: Overall, 5105 healthy indoor cats were tested and 8.1% (414/5105) had at least one subclinical infectious agent that is transmissible through blood product transfusion. 1.5% (77/5105) were positive for feline leukaemia virus antigens and 2.9% (148/5105) were positive for feline immunodeficiency virus antibodies, therefore they were excluded as donors. The overall prevalence of haemoplasmas in feline leukaemia virus and feline immunodeficiency virus SNAP-negative feline blood donors was 3.7% (181/4880) [1.3% (63/4880) were positive for Mycoplasma haemofelis; 2.3% (112/4880) to Candidatus Mycoplasma haemominutum and 0.12% (6/4880) to Candidatus Mycoplasma turicensis]. The prevalence of feline leukaemia provirus was 5.2% (9/173) and of Bartonella spp. was 0.2% (2/1122). None of the 173 cats were positive for Leishmania spp. CLINICAL SIGNIFICANCE: The prevalence of many transfusion transmissible pathogens was relatively high in this healthy, client-owned, indoor cats eligible to become blood donors. Performing an extended screening panel that includes at least polymerase chain reactions for Mycoplasma haemofelis, Candidatus Mycoplasma haemominutum, Candidatus Mycoplasma turicensis, feline leukaemia virus provirus, and Bartonella spp., in addition to feline leukaemia virus antigens and feline immunodeficiency virus antibodies, is recommended in pet blood banks from analogous regions.

Ménétrier's disease : a case of successful treatment using long-acting octreotide.

The authors describe a 31 years old male, admitted for hematemesis, epigastric pain and lower limb edema. Laboratorial data showed haemoglobin 18.4g/dl, total proteins 2.8g/dl, albumin 1.6g/dl and hipogammaglobulinaemia. 24h urinary proteins were normal. HIV and CMV serology were negative. Upper GI endoscopy revealed markedly enlarged gastric folds covered by abundant exudative fluid. Endoscopic ultrasound showed ascites, pleural effusion and gastric wall thickening with mucosa expansion and intact submucosa. In gastric biopsies foveolar hyperplastic and regenerative mucosa were observed being suggestive of Ménétrier´s disease. Helicobacter pylori was not detected. Albumin replacement and diuretics corrected anasarca and long-acting octreotide was instituted. Nine months later, the patient was asymptomatic, serum proteins were normal (albumin 4.6g/dl and total proteins 6.5g/dl), signs of endoscopic improvement were observed with marked reduction in gastric folds and mucosal inflammation and no ultrastructural changes were detected in gastric specimens sent for electron microscopy. Ménétrier´s Disease (MD) is a rare form of hypertrophic gastropathy characterized by massive enlargement of gastric folds causing marked protein exudation. The increase in tight junction diameter is the most consistent ultraestrutural change. Octreotide is a somatostatin analogue that acts by modulating TGFαEGFR pathway, which has been associated with the pathogenic mechanisms. As well as other cases reported in literature, this case report highlights the potential of long-acting octreotide for MD treatment avoiding more expensive therapies like cetuximab and gastrectomy.

Incidence, characteristics and geographical distributions of canine and human non-Hodgkin's lymphoma in the Porto region (North West Portugal).

Lymphoma is one of the most common neoplasms in dogs and it is one of the top five causes of cancer-related deaths, similar to human lymphoma. Companion animal epidemiological studies define dogs as sentinels of potential risk factors for human health, mainly due to shared environments, shorter disease latencies, and spontaneous disease. The aims of this study were to describe human and canine epidemiologic features of non-Hodgkin's lymphoma (NHL) and their similarities, and to investigate a possible geographical association in the incidence risks in the Greater Porto area, in north-western Portugal. The postal codes of human NHL patients diagnosed between 2005 and 2010 residing in the Greater Porto, Portugal, were obtained from North and Central Region Cancer Registries of Portugal. Available data from dogs diagnosed with lymphoma between 2005 and 2016 from several veterinary centres were also collected. Descriptive epidemiology, mapping cases, and age-standardised risks of NHL incidence (ASR) were determined for both species. The results showed a higher risk (P<0.05) of NHL in men (ASR men: 18.1 cases/100,000 inhabitants; women: 14.2 cases/100,000 inhabitants) and in male dogs (ASR males: 82 cases/100,000 dogs; females: 70 cases/100,000 dogs). The geographical distribution of human and canine ASR was well correlated (r=0.664, P<0.05), with the highest values for human and canine ASR detected in the same urban municipalities of the Greater Porto: Porto, Matosinhos and Maia. These findings suggest the existence of exposure similarities, supporting the relevance of cancer surveillance in pet animals as efficient tools to predict health hazards for humans.

A morphoanatomical approach to the adaptive features of the epidermis and proboscis of a marine Polychaeta: Eulalia viridis (Phyllodocida: Phyllodocidae).

Eulalia viridis is a marine Polychaeta of the rocky intertidal that, despite its simple anatomy, is an active predator of much larger invertebrates, from which it extracts pieces of soft tissue through suction. This uncanny feeding strategy triggered the pursuit for the morphological mechanisms that enable adaptation to its environment. The evaluation of the worm anatomy and microanatomy, combining electron and optical microscopy, revealed a series of particular adaptations in the epidermis and in the proboscis (the heavily muscled eversible pharynx). Besides its function in feeding, the proboscis is the main sensory organ, being equipped with numerous sensorial papillae holding chemoreceptors. Additionally, the proboscis possesses tentacles that become exposed when the organ is everted. These provide fast release of mucus and toxins, from mucocytes and special serous cells, respectively (the latter involving both merocrine and apocrine processes), whenever contact with a prey occurs. In its turn, the epidermis provides protection by cuticle and mucus secretion and has a sensorial function that may be associated to the worm's uncommon green pigment cells. Eulalia viridis presents a series of elegant adaptive tools to cope with its environment that are evolutionarily designed to counterbalance its relatively simple body plan.

Immunocytochemical study of canine lymphomas and its correlation with exposure to tobacco smoke.

AIM: Canine lymphoma is one of the most common canine neoplasms, but little is known regarding the effects of exposure to tobacco smoke on their biologic behavior. As cytology is the most frequent diagnostic method of canine lymphoma, the aims of this study were to perform an immunocytochemical study of canine lymphomas, including subtyping and cell proliferation analysis, and to establish their correlation with tobacco smoke exposure. MATERIALS AND METHODS: A total of 23 dogs diagnosed with lymphoma were subjected to careful fine-needle biopsies of enlarged lymph nodes. The smears were air-dried, fixed with cold acetone, and immunocytochemically stained using CD3, PAX5, and Ki-67. Owners were requested to complete an epidemiologic questionnaire. RESULTS: According to the updated Kiel classification, 65% were B-cell lymphomas - three low grade (LG) and 12 high grade (HG) and 35% were T-cell -two LG and six HG. Thirteen tumors presented high Ki67 indexes (>40%) (11 HG and 2 LG), two revealed moderate ones (20-40%) (1 HG and 1 LG), and three had low indexes (≤20%) (1 HG and 2 LG). Both a significant positive correlation and a significant linear-by-linear association (p=0.018) were observed between high Ki67 indexes and smoking owners (r=0.753, p=0.002) as well as with the number of smokers in the household (r=0.641, p=0.001). Moreover, the mean percentage of Ki67+ cells from the group of "smoker owners" was statically higher (p=0.011) than that from the "non-smoker owners." CONCLUSION: The results suggest that cytological diagnosis of canine lymphomas benefits from being complemented with immunocytochemical studies that include subtyping and assessment of proliferative activity, both contributing for the prognosis and therapeutic planning. Furthermore, exposure to tobacco smoke seems to be related to the biological behavior of canine lymphomas.

Cdc2-like kinase 2 in the hypothalamus is necessary to maintain energy homeostasis.

OBJECTIVE: To investigate whether the Cdc2-like kinase 2 (CLK2) is expressed in hypothalamic neurons and if it is, whether the hypothalamic CLK2 has a role in the regulation of energy balance. SUBJECTS: Swiss mice on chow or high-fat diet (HFD) and db/db mice on chow diet were used to address the role of CLK2 in the hypothalamus. RESULTS: Hypothalamic CLK2Thr343 phosphorylation, which induces CLK2 activity, is regulated in vivo by refeeding, insulin and leptin, in a PI3K (phosphoinositide 3-kinase)-dependent manner. The reduction of CLK2 expression in the hypothalamus, by chronic pharmacological inhibition with TG003 or by chronic knockdown with small interfering RNA was sufficient to abolish the anorexigenic effect of insulin and leptin, to increase body weight, fat mass, food intake and to decrease energy expenditure in mice on chow. In contrast, CLK2Thr343 phosphorylation in the hypothalamus in response to insulin, leptin or refeeding was impaired in mice on HFD or in db/db mice. Chronic CLK2 inhibition in the hypothalamus was associated with a slight increase in the fasting blood glucose levels, reduction in PEPCK (phosphoenolpyruvate carboxykinase) expression in the liver and enhanced glucose production from pyruvate, suggesting a regulation of hepatic glucose production. Further, overexpressing CLK2 in the mediobasal hypothalami of mice on HFD or in db/db mice by adenovirus partially reversed the obese phenotype. CONCLUSIONS: Thus, our results suggest that protein CLK2 integrates some important hypothalamic pathways, and may be a promising molecule for new therapeutic approaches for obesity and diabetes.

Comparison of simple sucrose and percoll based methodologies for synaptosome enrichment.

Synaptosomes are isolated nerve terminals. They represent an extremely attractive in vitro model system to study synaptic physiology since they preserve morphological and functional characteristics of the synapse. As such they have been used to investigate synaptic dysfunctions associated with neuropathologies like Alzheimer's disease. In the present work two simple methodologies for isolating synaptosomal-enriched fractions were compared for the first time. The starting points of both protocols were rat cortical or hippocampal homogenized tissues that underwent several differential centrifugation steps followed by a final purification of synaptosomal-enriched fractions using either a Percoll gradient or a Sucrose gradient. Comparison of the fractions obtained was carried out, using both biochemical and electron microscopy approaches. In the biochemical analysis the protein levels of pre-synaptic, post-synaptic, nuclear and mitochondrial markers were evaluated. Additional characterization of the synaptosomal-enriched fractions was performed using transmission electron microscopy. In summary, the results indicate that under the conditions tested the Sucrose based protocol is more efficient for the isolation of synaptosomal-enriched fractions from both neuronal tissues, being particularly efficient for hippocampus that is a less abundant brain tissue. Further, the sucrose protocol apparently results in a higher yield of viable synaptosomes suitable for further assays, including structural and functional studies of synapses; making this an attractive procedure to study processes associated with neuropathologies.

RNA sequencing reveals region-specific molecular mechanisms associated with epileptogenesis in a model of classical hippocampal sclerosis.

We report here the first complete transcriptome analysis of the dorsal (dDG) and ventral dentate gyrus (vDG) of a rat epilepsy model presenting a hippocampal lesion with a strict resemblance to classical hippocampal sclerosis (HS). We collected the dDG and vDG by laser microdissection 15 days after electrical stimulation and performed high-throughput RNA-sequencing. There were many differentially regulated genes, some of which were specific to either of the two sub-regions in stimulated animals. Gene ontology analysis indicated an enrichment of inflammation-related processes in both sub-regions and of axonal guidance and calcium signaling processes exclusively in the vDG. There was also a differential regulation of genes encoding molecules involved in synaptic function, neural electrical activity and neuropeptides in stimulated rats. The data presented here suggests, in the time point analyzed, a remarkable interaction among several molecular components which takes place in the damaged hippocampi. Furthermore, even though similar mechanisms may function in different regions of the DG, the molecular components involved seem to be region specific.

Pioglitazone treatment increases food intake and decreases energy expenditure partially via hypothalamic adiponectin/adipoR1/AMPK pathway.

INTRODUCTION: Thiazolidinediones (TZDs) enhanced body weight (BW) partially by increased adipogenesis and hyperphagia. Neuronal PPARγ knockout mice on high-fat diet (HFD) are leaner because of enhanced leptin response, although it could be secondary to their leanness. Thus, it still is an open question how TZDs may alter energy balance. Multiple factors regulate food intake (FI) and energy expenditure (EE), including anorexigenic hormones as insulin and leptin. Nonetheless, elevated hypothalamic AMPK activity increases FI and TZDs increase AMPK activity in muscle cells. Thus, the aim of the present study was to investigate whether Pioglitazone (PIO) treatment alters hypothalamic insulin and leptin action/signaling, AMPK phosphorylation, and whether these alterations may be implicated in the regulation of FI and EE. METHODS: Swiss mice on HFD (2 months) received PIO (25 mg kg(-1) per day-gavage) or vehicle for 14 days. AMPK and AdipoR1 were inhibited via Intracerebroventricular injections using Compound C (CompC) and small interference RNA (siRNA), respectively. Western blot, real-time PCR and CLAMS were done. RESULTS: PIO treatment increased BW, adiposity, FI, NPY mRNA and decreased POMC mRNA expression and EE in HFD mice. Despite higher adiposity, PIO treatment improved insulin sensitivity, glucose tolerance, decreased insulin and increased adiponectin serum levels. This result was associated with, improved insulin and leptin action/signaling, decreased α2AMPK(Ser491) phosphorylation and elevated Acetyl-CoA carboxylase and AMPK(Thr172) phosphorylation in hypothalamus. The inhibition of hypothalamic AMPK with CompC was associated with decreased adiposity, FI, NPY mRNA and EE in PIO-treated mice. The reduced expression of hypothalamic AdipoR1 with siRNA concomitantly with PIO treatment reverted PIO induced obesity development, suggesting that adiponectin may be involved in this effect. CONCLUSIONS: These results demonstrated that PIO, despite improving insulin/leptin action in hypothalamus, increases FI and decreases EE, partially, by activating hypothalamic adiponectin/AdipoR1/AMPK axis. Suggesting a novel mechanism in the hypothalamus by which TZDs increase BW.

Prevalence of antimicrobial resistance in faecal enterococci from vet-visiting pets and assessment of risk factors.

The objective of this study was to determine the prevalence of antimicrobial resistance (AMR) exhibited by enterococci isolated from faeces of pets and its underlying risk factors. From September 2009 to May 2012, rectal swabs were collected from 74 dogs and 17 cats, selected from the population of animals visiting the Veterinary Hospital of University of Porto, UPVet, through a systematic random procedure. Animal owners answered a questionnaire about the risk factors that could influence the presence of AMR in faecal enterococci. Enterococci isolation, identification and antimicrobial (AM) susceptibility testing were performed. Data analyses of multilevel, univariable and multivariable generalised linear mixed models were conducted. From all enterococci isolated (n=315), 61 per cent were considered multidrug-resistant, whereas only 9.2 per cent were susceptible to all AMs tested. Highest resistance was found to tetracycline (67.0 per cent), rifampicin (60.3 per cent), azithromycin (58.4 per cent), quinupristin/dalfopristin (54.0 per cent) and erythromycin (53.0 per cent). Previous fluoroquinolone treatments and coprophagic habits were the features more consistently associated with the presence of AMR for three (chloramphenicol, ciprofloxacin and azithromycin) and seven (tetracycline, rifampicin, gentamicin, chloramphenicol, ciprofloxacin, erythromycin and azithromycin), respectively, out of nine AMs assessed. Evaluating risk factors that determine the presence of drug-resistant bacteria in pets, a possible source of resistance determinants to human beings, is crucial for the selection of appropriate treatment guidelines by veterinary practitioners.

Interobserver Reproducibility of Histological Grading of Canine Simple Mammary Carcinomas.

Histological grading of canine mammary carcinomas (CMCs) has been performed using an adaptation of the human Nottingham method. The histological grade could be a prognostic factor in CMC; however, no data are available concerning interobserver variability in grading. In this study we analyzed the interobserver reproducibility between three observers when assigning individual parameter scores and grade to 46 CMCs. The influence of tumour size and vascular invasion and/or lymph node metastases on the odds of grading disagreement was also evaluated. The mean kappa values were 0.71, 0.51, 0.69 and 0.70 for tubule formation, nuclear pleomorphism, mitotic counts and grade, respectively. There was moderate to good agreement in scoring parameters and tumour grading, with nuclear pleomorphism being least reproducible. These findings are similar to those of human studies. The odds of grading disagreement increased with tumour size, but decreased with the presence of vascular invasion and/or lymph node metastases. Individual scoring differences were moderated by reaching a consensus between two observers.

Molecular characterization of quinolone resistance mechanisms and extended-spectrum ß-lactamase production in Escherichia coli isolated from dogs.

The increasing prevalence of antimicrobial resistances is now a worldwide problem. Investigating the mechanisms by which pets harboring resistant strains may receive and/or transfer resistance determinants is essential to better understanding how owners and pets can interact safely. Here, we characterized the genetic determinants conferring resistance to ß-lactams and quinolones in 38 multidrug-resistant Escherichia coli isolated from fecal samples of dogs, through PCR and sequencing. The most frequent genotype included the ß-lactamase groups TEM (n=5), and both TEM+CTX-M-1 (n=5). Within the CTX-M group, we identified the genes CTX-M-32, CTX-M-1, CTX-M-15, CTX-M-55/79, CTX-M-14 and CTX-M-2/44. Thirty isolates resistant to ciprofloxacin presented two mutations in the gyrA gene and one or two mutations in the parC gene. A mutation in gyrA (reported here for the first time), due to a transversion and transition (TCG→GTG) originating a substitution of a serine by a valine in position 83 was also detected. The plasmid-encoded quinolone resistance gene, qnrs1, was detected in three isolates. Dogs can be a reservoir of genetic determinants conferring antimicrobial resistance and thus may play an important role in the spread of antimicrobial resistance to humans and other co-habitant animals.

Development of multicore hybrid particles for drug delivery through the precipitation of CO2 saturated emulsions.

Hybrid lipid-polymer particles are gaining increasing interest to be applied as drug delivery systems due to their greater stability in biological fluids and enhanced cellular uptake of drugs. However, a major drawback is the fact that these particles are usually produced through techniques that use organic solvents. This work studies the possibility of producing for the first time hybrid particles composed by lipid multicores enveloped in a polymeric layer through Particles from Gas Saturated Solutions (PGSS(®)), without using organic solvents. An oil-in-water emulsion, composed by Gelucire 43/01™ (GEL) as the discontinuous phase and by an aqueous polyethylene glycol 4000 (PEG) solution as the continuous phase, was successfully precipitated by PGSS(®). Operating conditions that ensured the stability of the CO2 saturated emulsion were previously evaluated. The resulting PEG-GEL particles present a spherical-like morphology constituted by several lipid cores encapsulated into a polymeric shell. The applicability of these structured particles to be used as drug delivery system (DDS) was studied by using ketoprofen, a nonsteroidal anti-inflammatory drug (NSAID), as model drug. The particles provided an initial burst release of the drug due to the progressive dissolution of the external layer of PEG, followed by a controlled release of the NSAID from the GEL cores.

Age-associated Epstein-Barr virus-specific T cell responses in seropositive healthy adults.

Epstein-Barr virus (EBV) is present in 95% of the world's adult population. The immune response participates in immune vigilance and persistent infection control, and this condition is maintained by both a good quality (functionality) and quantity of specific T cells throughout life. In the present study, we evaluated EBV-specific CD4(+) and CD8(+) T lymphocyte responses in seropositive healthy individuals younger and older than 50 years of age. The assessment comprised the frequency, phenotype, functionality and clonotypic distribution of T lymphocytes. We found that in both age groups a similar EBV-specific T cell response was found, with overlapping numbers of tumour necrosis factor (TNF)-α(+) T lymphocytes (CD4(+) and CD8(+)) within the memory and effector cell compartments, in addition to monofunctional and multi-functional T cells producing interleukin (IL)-2 and/or interferon (IFN)-γ. However, individuals aged more than 50 years showed significantly higher frequencies of IL-2-producing CD4(+) T lymphocytes in association with greater production of soluble IFN-γ, TNF-α and IL-6 than subjects younger than 50 years. A polyclonal T cell receptor (TCR)-variable beta region (Vß) repertoire exists in both age groups under basal conditions and in response to EBV; the major TCR families found in TNF-α(+) /CD4(+) T lymphocytes were Vß1, Vß2, Vß17 and Vß22 in both age groups, and the major TCR family in TNF-α(+) /CD8(+) T cells was Vß13·1 for individuals younger than 50 years and Vß9 for individuals aged more than 50 years. Our findings suggest that the EBV-specific T cell response (using a polyclonal stimulation model) is distributed throughout several T cell differentiation compartments in an age-independent manner and includes both monofunctional and multi-functional T lymphocytes.

Comparison of two hydrogel formulations for drug release in ophthalmic lenses.

In the present work two types of polymers were investigated as drug releasing contact lens materials: a poly-hydroxyethylmethacrylate (pHEMA) based hydrogel and a silicone hydrogel. The silicone hydrogel resulted from the addition of TRIS, a hydrophobic monomer containing silicon (3-tris(trimethylsilyloxy)silylpropyl 2-methylprop-2-enoate), to pHEMA. Both hydrogels were loaded with an antibiotic (levofloxacin) and an antiseptic (chlorhexidine) by soaking in the drug solutions. The hydrogel properties were determined to be within the range demanded for lens materials. The release profiles of both drugs from the hydrogels were obtained and eventual drug/polymer interactions were assessed with the help of Raman spectra. A mathematical model, developed to mimic the eye conditions, was applied to the experimental results in order to predict the in vivo efficacy of the studied systems. The release profiles were compared with those resulting from the application of commercial eyedrops. The pHEMA based hydrogel demonstrated to be the best material to achieve a controlled release of levofloxacin. In the case of chlorhexidine, the silicone hydrogel seems to lead to better results. In both cases, our results suggest that these materials are adequate for the preparation of daily disposable therapeutic contact lenses.

Genetic characterization of CCL3, CCL4 and CCL5 in leporid genera Oryctolagus, Sylvilagus and Lepus.

The genetic diversity of C-C motif chemokine receptor 5 (CCR5) ligands CCL3, CCL4 and CCL5 in the leporid genera Oryctolagus, Sylvilagus and Lepus was studied. Our results demonstrate that the three CCR5 chemokine ligands are under strong purifying selection as a result of possible functional binding constraints.